print version cee 697z organic compounds in water and ... · 24 feb 2010 1 ppcp analysis october...
TRANSCRIPT
24 Feb 2010
1
PPCP Analysis
October 27, 2014
Lecture given by Kaoru Ikuma, Ph.D.
CEE 697z - Lecture #21 1
CEE 697zOrganic Compounds in Water and Wastewater
Print version
EPA Method 1694 Pharmaceuticals
and personal care products in water, soil, sediment, and biosolids by HPLC/MS/MS
Four analytical groups
2CEE 697z - Lecture #21
24 Feb 2010
2
CEE 697z - Lecture #21 3
The Analytical Chain of LC/MS
Sample
Results
Sample Treatment
Separations
Data/Interpretation
Mass Analysis
Ionization/Interfaces
Step 1Sample treatment: Extraction
CEE 697z - Lecture #21 4
24 Feb 2010
3
Solid Phase Extraction
Extraction of organic contaminants from water and adsorb onto solid phase to concentrate
Sample pretreatment method used to quantitatively analyze contaminants with Liquid Chromatography/Mass Spectroscopy
Solid phase contained in cartridges or barrels
EluteCondition/Equilibrate
Load Wash
CEE 697z - Lecture #21 6
Extraction Technologies
Off-line Solid-Phase Extraction (liquids) Cartridges (syringe, sep-pak)
Disks
96-well plates
Solid-phase microextraction
On-line Solid-Phase Extraction (liquids) Prospekt cartridges
Accelerated Solvent Extraction (solids) Sorbents?
Slide courtesy of Meyer et al., USGS
24 Feb 2010
4
CEE 697z - Lecture #21 7
Solid-Phase Extraction
Many Types of Materials
C2-18 on Silica backbone with varying linkages
Polymers also with hydrophillic-lipophilic functional groups
Anion Exchange (WC, SC, WA, SA)
Mixed Mode
Immunnoaffinity
Many manufacturers
Slide courtesy of Meyer et al., USGS
CEE 697z - Lecture #21 8
SPE Example
100 mL sample
1 mg Na2EDTA
75 L of 40% H2SO4
60 mg HLBCondition: 2 mL MeOH
3 mL 0.5 N HCl1 mL H2O
Load Sample
Wash: 1 mL water
Elute: 5 mL MeOH into test tube
Concentrate: N2 to 125 L
Analyze: ESI(+)
Slide courtesy of Meyer et al., USGS
24 Feb 2010
5
CEE 697z - Lecture #21 9
Typical Concentration Factors:Environmental SPE
Sample Extract Concentration
Vol. (ml) Vol. (µL) Factor
100 100 1000
1,000 500-1000 1000-2000
1 µg/L 1-2 mg/L
Slide courtesy of Meyer et al., USGS
Off-line Manual SPE Method
500 mL to 1000 mL sample size concentrated to 1 mL
One-time use HLB extraction cartridges
6 hour extraction method time
16 min instrument run time
Prior to LC/MS/MS
24 Feb 2010
6
CEE 697z - Lecture #21 11
The Analytical Chain of LC/MS
Sample
Results
Sample Treatment
Separations
Data/Interpretation
Mass Analysis
Ionization/Interfaces
Step 2Separation: Liquid chromatography
CEE 697z - Lecture #21 12
24 Feb 2010
7
CEE 697z - Lecture #21 13
The Analytical Chain of LC/MS
Sample
Results
Sample Treatment
Separations
Data/Interpretation
Mass Analysis
Ionization/InterfacesSeparation
Alternatives
Reversed-Phase LCNormal Phase LCSize ExclusionIon Exchange LCCapillary Electrophoresis
CEE 697z - Lecture #21 14
What is Chromatography ?
http://www.micromountain.com/sci_diagrams/sci_app/sci_app_pages/ctography_lab_eng.htm
Stationary phase Mobile phase
24 Feb 2010
8
CEE 697z - Lecture #21 15
High performance liquid chromatography (HPLC)
http://en.wikipedia.org/wiki/Chromatography
HPLC Instrument Basics
MOBILEPHASE
PUMPINJECTION
POINT
RECORDER
DETECTOR
COLLECTOR
COLUMN
24 Feb 2010
9
Types of HPLC Phases
Adsorption Normal Phase – polar bed, non polar mobile
phase (n-hexane, tetrahydrofuran) Reverse Phase – non-polar bed w/ polar mobile
phase (methanol, water, acetonitrile mixture) * most common
Ion Exchange Stationary bed ionically charged surface,
opposite to sample ions Use with ionic or ionizable samples Stronger charge = longer elution time Mobile Phase – aqueous buffer
Size Exclusion Column material precise pore sizes Large molecules first, then small
CEE 697z - Lecture #21 17
Mobile Phase / Eluent
o All solvents “HPLC grade” Filtered using 0.2 μm filter Extends pump life Protects column from clogs
o Solvent Degassing / Purging Displacement w/ less soluble gas Vacuum application Heat solvent
- Purity - Low viscosity
- Detector compatibility - Chemical inertness
- Solubility of sample - Price
CEE 697z - Lecture #21 18
24 Feb 2010
10
CEE 697z - Lecture #21 19
HPLC Columns (stationary phase)
Stainless steel
Common sizes: 10,12.5, 15, 25 cm long
4.6 mm i.d.
Length for optimum separation dictated by theoretical plates needed for good resolution
Filled with stationary phase material (typically particles of ~5 μm)
CEE 697z - Lecture #21 20
24 Feb 2010
11
CEE 697z - Lecture #21 21
HPLC vs. UPLC (ultra performance LC)
Thakkar, et al., 2011
Aripiprazole Dopamine agonist, used as a second generation antipsychotic drug
HPLC
UPLC
CEE 697z - Lecture #21 22
What is LC/MS/MS ?
It is Liquid chromatography coupled with Mass Spectrometer
The discussion is restricted to the available instrument by Waters, Milford, MA (Micromass Quattro micro API Mass Spectrophotometer)
24 Feb 2010
12
CEE 697z - Lecture #21 23
Power of LC/MS/MS
MS provides exceptionally clean product (fragment) ion chromatograms for quantification
The signal-to-noise (S/N) ratio is optimized
Useful for the rapid screening of complex samples where analytes of interest are known
Compound identity confirmation can be achieved with MS/MS using the product ion scan mode
By detecting a specific product ion (precursor ion mode) or charged fragments resulting from a neutral loss (neutral loss mode), you can classify a compound of interest
CEE 697z - Lecture #21 24
General Principle of Operation of LC/MS/MS
http://en.wikipedia.org/wiki/Lipidomics
24 Feb 2010
13
CEE 697z - Lecture #21 25
The Analytical Chain of LC/MS
Sample
Results
Sample Treatment
Separations
Data/Interpretation
Mass Analysis
Ionization/Interfaces
Step 3Interface and ionization
CEE 697z - Lecture #21 26
24 Feb 2010
14
CEE 697z - Lecture #21 27
The Analytical Chain of LC/MS
Sample
Results
Sample Treatment
Separations
Data/Interpretation
Mass Analysis
Ionization/Interfaces
Ionization/InterfacesAlternatives
ElectrosprayAtmospheric Pressure CIParticle BeamContinuous Flow FABThermosprayInductively Coupled
Plasma
CEE 697z - Lecture #21 28
Interface and Ionization
All interface/ionization combination must convert dissolved analyteeluting from a separation system into gas-phase ions at reduced pressure.
State-of-matter: Liquid-phase Evaporation Gas Phase
Pressure: Atmospheric Pressure Reduction High Vacuum
Charge State: Neutral(Ionic) Ionization Ionic
LC Conversion Process MS
24 Feb 2010
15
CEE 697z - Lecture #21 29
Ionization Source
Broad range of atmospheric pressure ionization (API) sources
Electrospray (ESI) probe – the most widely used API technique for sensitive, general analysis of polar & ionic comp.
Atmospheric Pressure Chemical Ionization (APCI) probe –ionization capabilities for less polar & neutral chemical species
IonSABRE™ APCI – excellent sensitivity for less polar & nonpolar analytes, especially at higher liquid flow rates
ESCi™ Multi-Mode Ionization – combines ESI and APCI in the same analysis
APPI™/APCI Dual Ionization – provides APCI in simultaneous operation with photoionization (PI)
MUX-technology™ – provides the ability to multiplex four sample streams into a single Waters Micromass mass spectrometer
Ionization-Continuum Diagram
CEE 697z - Lecture #21 30Slide courtesy of Meyer et al., USGS
24 Feb 2010
16
CEE 697z - Lecture #21 31
Electrospray (ES)
In an electrospray interface, the column effluent of LC is nebulized into an atmosphere-pressure ion source.
ES is composed of a hollow needle with a high electrical potential through which the effluent flows (1-10uL/min).
The high field at the tip of the needle produces a cone shaped liquid meniscus from which a spray of highly charged droplets emerges.
Subsequent evaporation of the droplets results in ion formation.
Ionization in Electrospray
Ionization of the solute in solution.
Nebulize the solution and charge the droplets.
Desolvation of the droplets by evaporation.
Desorption of the solution ions to gas phase ions.
CEE 697z - Lecture #21 32
Slide courtesy of Meyer et al., USGS
24 Feb 2010
17
CEE 697z - Lecture #21 33
From: Harris, 1999
ESI types
Positive Use volatile proton donor (e.g., 0.1% formic
acid)
Negative Use volatile proton acceptor (e.g., 0.3%
NH4OH)
CEE 697z - Lecture #21 34
24 Feb 2010
18
Matrix Effects
Suppression
Enhancement
Mostly occur in ESI
CEE 697z - Lecture #21 35
CEE 697z - Lecture #21 36
The Analytical Chain of LC/MS
Sample
Results
Sample Treatment
Separations
Data/Interpretation
Mass Analysis
Ionization/Interfaces
24 Feb 2010
19
Step 4Mass analysis: Mass spectrometer
CEE 697z - Lecture #21 37
CEE 697z - Lecture #21 38
The Analytical Chain of LC/MS
Sample
Results
Sample Treatment
Separations
Data/Interpretation
Mass Analysis
Ionization/Interfaces Mass AnalysisAlternatives
QuadrupoleIon TrapTime-of-FlightSectorFourier Transform
24 Feb 2010
20
CEE 697z - Lecture #21 39
Types of MS
4 Types commonly used in environmental analysis Magnetic Sector MS
Quadrupole MS
Ion-trap MS
Time of Flight MS
Others Fourier Transform Ion Cyclotron Resonance MS
(FT-ICR)
CEE 697z - Lecture #21 40
MS Quadrupole
Most common mass analyzer in use since the 1950s
Quadrupole MS are smaller, cheaper and more rugged than magnetic sectors
Low scan times (<100 ms) – ideal for GC or LC inlets Called mass filters rather than mass analyzers
ions of only a single mass to charge (m/q) ratio pass through the apparatus
separate ions based on oscillations in an electric field (the quadrupole field) using AC and DC currents
24 Feb 2010
21
CEE 697z - Lecture #21 41
Schematic of Quadrupole
Hardy, U of Akron
CEE 697z - Lecture #21 42
Operation of Quadrupole Mass Filter
voltages applied to electrodes affect trajectory of ions with the m/q ratio of interest as they travel down the center of the four rods
these ions pass through the electrode system
ions with other m/z ratios are thrown out of their original path
these ions are filtered out or lost to the walls of the quadrupole, and then ejected as waste by a vacuum system
in this manner the ions of interest are separated
24 Feb 2010
22
MS/MS
Quadrupole
CEE 697z - Lecture #21 43
Precursor ion Product ion(s)fragmentation
CEE 697z - Lecture #21 44
Time-of-Flight Mass Spectrometry
Ionization: positive ions are produced periodically by bombardment of the sample with brief pulses of electrons, secondary ions, or in cases laser-generated photons.
Acceleration: The ions are then accelerated by an electric field pulse of 103 to 104 V (the “pusher”) that has the same frequency as, but lags behind, the ionization pulse
Drift: The accelerated particles then pass into a field-free drift tube. The drift tube’s length can range from 0.5 - 3.0 meters
24 Feb 2010
23
CEE 697z - Lecture #21 45
Lighter ions are subject to greater acceleration
Time-of-flight MS
CEE 697z - Lecture #21 46
Xevo G2-XS QTof
24 Feb 2010
24
CEE 697z - Lecture #21 47
The Analytical Chain of LC/MS
Sample
Results
Sample Treatment
Separations
Data/Interpretation
Mass Analysis
Ionization/Interfaces
Step 5Data analysis and interpretation
CEE 697z - Lecture #21 48
24 Feb 2010
25
Exact Mass, MS/MS for DEET
CEE 697z - Lecture #21 49
From: Schreiber et al., 2010
MS/MS Transitions for DEET
CEE 697z - Lecture #21 50
From: Schreiber et al., 2010
24 Feb 2010
26
QA/QC of LC/MS/MS-based PPCP analysis
CEE 697z - Lecture #21 51
Experimental design
Method detection limit (MDL)
Analytical sensitivity
Calibration drift
% Recovery
CEE 697z - Lecture #21 52
24 Feb 2010
27
Typical MDLs and % recoveries
CEE 697z - Lecture #21 53
By LC/MS/MS (API 4000 triple quadrupole MS)
Wilcox et al., 2009 JEQ
What is causing the lower % recoveries?
CEE 697z - Lecture #21 54
The Analytical Chain of LC/MS
Sample
Results
Sample Treatment
Separations
Data/Interpretation
Mass Analysis
Ionization/Interfaces
24 Feb 2010
28
Bioassays
CEE 697z - Lecture #21 55
Biological Activity Tests
Estrogenic and antiestrogenic activity will be assessed by measuring changes in gene expression in the Japanese medaka fish. expose fish to 1-L water sample for 96 hours
sacrifice the fish; livers removed other tissues, e.g. gonads and brain also will be removed, stored in RNAlater
and archived for potential future studies or examination of expression of other genes
measure vitellogen in mRNA in the liver using real time reverse transciptase PCR (Roche Light Cycler). Detection limit is typically 10 femtomolar
Vitellogenin, the precursor egg yolk protein normally produced only in female fish - but
male fish exposed to xenoestrogens will also produce it.
CEE 697z - Lecture #21 56
24 Feb 2010
29
Routledge, E.J. and Sumpter, J.P. (1996) Environ. Toxicol. Chem. 15, 241-248
The Yeast Estrogen Screen (YES) is an in-vitro test to measure estrogenic activity
Erik Rosenfeldt
CEE 697z - Lecture #21 57
Compound EC50 (nM) E2eq
E2 ~0.21 1.0
E1 ~0.3 0.7
EE2 ~0.20 1.1
NP ~1050 0.0003
Activity Assay Outputs
YES Calibration Curves
MIC Calibration Curves
Compound 80% Inhibition (mg/L)
SMX 10 (max 82%)
SDZ ~15 (max 83%)
SMZ ~102 (max 82%)
TMP ~0.15 (max 100%)
Compound EC50 (nM)
E2 ~0.21
E1 ~0.3
EE2 ~0.20
NP ~1050Erik Rosenfeldt
24 Feb 2010
30
Antibiotic Challenge (ABC)
Relies upon growth of Bacillus stearothermophilusspores
Test takes 2.5 hours
Negative PositiveErik Rosenfeldt
ABC Calibrations
Pennicillin Calibration in DIAmoxicillin Calibration
in DI
Pennicillin Calibration in Natural Water
y = 3.9578xR² = 0.947
y = 3.648xR² = 0.9935
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.05 0.1 0.15 0.2 0.25
Fra
ctio
nal
Res
po
nse
Disolved Penicillin (ug/L)
Penicillin in DI
Penicillin in Natural Water
Pennicillin Calibration Curves (Linear Region)
Erik RosenfeldtCEE 697z - Lecture #21 60
24 Feb 2010
31
CEE 697z - Lecture #21 61
Immunoassays
Slide courtesy of Meyer et al., USGS
CEE 697z - Lecture #21 62
Immunoassay Types
Enzyme-Linked Immunosorbent Assays
Coated
Tubes
96-well plate
Magnetic Particle
Radioimmunoassay
H3, C14
Slide courtesy of Meyer et al., USGS